Surgical trauma produces a profound inflammatory response that, when deregulated, leads to adverse surgical outcomes including protracted recovery, infection, and organ dysfunction. The immune response to surgery involves complex, multi-cellular mechanisms that are poorly understood. The long-term goals of this proposal are to: 1) use a systems-wide approach to enumerate and characterize the major immune cell subsets, on a cell-by-cell basis, from whole blood samples taken from patients undergoing surgery; 2) understand the mechanistic basis of immune-modulatory interventions designed to improve surgical outcomes (e.g., L-arginine supplementation); 3) understand the interplay between innate and adaptive immunity in order to identify specific mechanisms that are critical for patients' recovery from surgery. To achieve these goals, Dr. Gaudilliere will use a next-generation flow cytometry platform (Cytometry by Time of Flight or CyTOF), recently pioneered and brought to practical utility in the laboratory of Dr. Garry Nolan (Professor of Microbiology and Immunology, Stanford University and primary mentor for this K23 award). Uniting flow cytometry with mass spectrometry enables readouts from rare earth metal isotopes tagged to antibodies. In contrast to traditional fluorescence-based cytometry, the absence of overlap between detection signals allows for a dramatic increase in the number of parameters that can be measured at the single-cell level (currently up to 45). In a pilot study and working under the guidance of Drs. Garry Nolan and Martin Angst (Professor of Anesthesiology, Stanford University), Dr. Gaudilliere established a quantitative and reproducible mass cytometry assay with which to monitor the immune response in patients undergoing hip replacement (i.e., total hip arthroplasty). The data from this study form the groundwork for Dr. Gaudilliere's core hypothesis that Surgery-induced Myeloid Cells (SiMCs) phenocopy MDSCs and suppress the CD8+ T cell adaptive immune response to surgery via an L-arginine-dependent mechanism. In Aim 1, Dr. Gaudilliere will build an in vitro system to investigate whether SiMCs suppress terminal effector CD8+ T cell (CD8+Teff) function via an L- arginine-dependent mechanism. In Aim 2, Dr. Gaudilliere proposes a first interventional clinical trial that will use mass cytometry o investigate whether L-arginine supplementation in patients undergoing THA will restore CD8+Teff response in vivo. In Aim 3, Dr. Gaudilliere will adapt and implement statistical tools and learning algorithms (e.g., the least absolute shrinkage and selection operator or LASSO) to investigate whether patient- specific immune features predict surgery-induced expansion of SiMCs and suppression of CD8+Teff cells. Dr. Gaudilliere is an anesthesiologist at Stanford University School of Medicine with a background in engineering, biochemistry, and molecular biology, and is therefore exceptionally well qualified to address these aims. The Nolan Lab, acknowledged as world-class in the application of mass cytometry to single-cell analysis, will provide Dr. Gaudilliere with the opportunity and environment to acquire the skills for him to become a leading expert in this technology. Furthermore, Dr. Gaudilliere is supported by a multidisciplinary and collaborative team with expertise in signaling biology, human immunology, statistics and bio-informatics, and clinical experimental science and trial design. He will also benefit from the combined strength and resources provided by the Stanford Departments of Anesthesiology, Immunology, and Statistics. To accomplish his research goals and prepare him for a career as an independent investigator, Dr. Gaudilliere has created a multi-disciplinary career development plan incorporating: 1) advanced training in human immunology and immune monitoring with mass cytometry; 2) graduate level didactics in epidemiology and mentored training in clinical study design; and 3) graduate level didactics and mentored training in biostatistics, data mining, and application of machine learning methods for the analysis of complex datasets derived from mass cytometry. In summary, single-cell mass cytometry will be utilized to monitor immune responses to surgery at the systems level in vivo. This approach will not only elucidate specific mechanisms (e.g., arginine-dependent SiMC-mediated suppression of CD8+ T cells) but will also characterize these mechanisms as they occur in the context of the entire immune system. The multidimensional attribute of the data will necessarily generate deeper and potentially more clinically relevant hypotheses than previously posed. The output of this proposal constitutes a data-driven strategy to guide future research efforts and R01 applications to identify patient- specific immune traits predictive of surgical outcomes and explore novel immune-modulatory strategies to improve recovery from surgery.